Composition and uses thereof

ABSTRACT

The present invention provides a particle comprising a fusion protein, wherein the fusion protein comprises at least one NANP repeat (SEQ ID NO: 7), some or all of the C-terminus of the CS protein from  Plasmodium falciparum  and a hepatitis B surface antigen, and wherein the particle comprises no, or substantially no, free hepatitis B surface antigen protein, and uses thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation Application of U.S. application Ser.No. 14/762,106, filed Jul. 20, 2015, which is a 35 U.S.C. §371 NationalPhase Entry Application of International Application No.PCT/GB2014/050156, filed Jan. 21, 2014, which designates the U.S., andwhich claims the benefit of GB Application No. 1308242.5, filed May 8,2013, and the benefit of GB Application No. 1301022.8, filed Jan. 21,2013, the contents of each of which are incorporated herein by referencein their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Oct. 20, 2015 isnamed 20151022_Sequence_Listing_052342-085440-US.txt and is 15,217 bytesin size.

The present invention relates to immunogenic compositions for use ineliciting immune responses to pathogenic organisms, and in particular,for the prevention of malaria. The invention also provides for the useof the immunogenic compositions, in particular, for use in eliciting aprotective immune response.

Plasmodium falciparum malaria is one of the major infectious diseases ofmankind. It is the most important parasitic cause of human mortality anda major cause of death in young African children. Current estimates ofannual deaths from malaria vary from about 700,000 to over a million.Most of this mortality is in Africa. Additionally there are about 250million clinical cases of malaria each year, providing a huge burden ofmorbidity that adds to the disease burden caused by malaria deaths.Although current control measures, such as bed nets, insecticidespraying and rapid drug treatments are partially effective it is widelyconsidered important to develop new tools to control malaria. Probablythe most useful of these tools would be an effective malaria vaccine.

Scientists have been attempting to develop a malaria vaccine for over ahundred years (Sergent & Sergent C. R. Acad. Sci. 151:407-409. 1910) andprogress has been slow. The most advanced vaccine in clinicaldevelopment is called RTS,S which has been in clinical development forover 15 years (Gordon et al. J Infect Dis 1995).

The RTS,S vaccine was developed by GSK Biologicals from 1988 to anongoing phase III trial in African children. This vaccine induces highantibody responses that bind to the major surface component of themalaria sporozoite, the circumsporozoite protein (CS protein), andthereby prevent or reduce parasite entry into the liver (Kester, K. E.,et al. J Infect Dis 200, 337-346, 2009). The vaccine is an unusualconstruct. It comprises a fusion protein of most of the circumsporozoiteprotein fused at the gene level to the DNA encoding the surface antigenof hepatitis B (HBsAg). This “RTS” (R=repeat, T=T cell epitopecontaining C-terminus, S=hepatitis B surface antigen) component isco-expressed in the yeast Saccharomyces cerevisiae with a substantialexcess of hepatitis B surface antigen (hence RTS, S) with a stochiometryof one part of RTS to four parts of S. This results in only about 14% ofRTS,S by mass being part of the P. falciparum CS protein. The excess ofS was required to allow RTS to form particles and in turn enhance theimmunogenicity of the R component of the vaccine. The generation of suchsmall particles, of about 23 nm in size, often called virus-likeparticles, is known to be key to enhancing the immunogenicity of manyvaccine antigens. However, the disadvantage of this RTS,S particle isthat a large part of the immune response, in particular the antibodyresponse, is induced to hepatitis B and only a minority to malaria.Nonetheless, this is the most effective single component vaccine evertested for malaria and when combined with the saponin+MPL+liposomalformulation adjuvant AS01 reliably induces about 45% sterile efficacy insporozoite challenge studies (Kester, K. E., et al. J Infect Dis 200,337-346, 2009), with similar levels of efficacy (39% reduction inepisodes over 12 months of follow-up) in a large phase III) study of5-17 month old children in Tanzania and Kenya (Olotu, A., et al. LancetInfect Dis 11, 102-109, 2011).

An aim of the present invention is to provide an alternative immunogenfor use in the prevention of malaria, wherein the efficacy of theimmunogen is preferably greater than RTS,S.

According to a first aspect the invention provides a particle comprisinga fusion protein comprising at least one NANP repeat (SEQ ID NO: 7),some or all of the C-terminus of the CS protein from Plasmodiumfalciparum and a hepatitis B surface antigen.

Preferably the hepatitis B surface antigen is the S antigen.

The NANP repeat (SEQ ID NO: 7) is a repeat of the four amino acidsasparagine, alanine, asparagine, proline which occurs naturally in theCS protein from Plasmodium falciparum.

There may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18 or more repeats of NANP (SEQ ID NO: 8). Preferably there are at least3 repeats, more preferably there are at least 10 repeats. The fusionprotein may in one embodiment comprise 18 repeats of NANP (SEQ ID NO:9).

The C-terminus of the CS protein is often referred to as the T-cellepitope containing C-terminus. The C-terminus of the CS protein includedin the fusion protein of the invention may comprise the sequence:NKNNQGNGQGHNMPNDPNRNVDENANANSAVKNNNNEEPSDKHIKEYLNKIQNSLSTEWSPCSVTCGNGIQVRIKPGSANKPKDELDYANDIEKKICKMEKCSSVFNVVNSSIGI (SEQ ID NO:6) with some of the C-terminal amino acids deleted. Preferably up to 15amino acids are deleted, more preferably up to 10 amino acids, 9 aminoacids, 8 amino acid, 7 amino acids, 6 amino acids, 5 amino acids, 4amino acids, 3 amino acids are deleted.

The C-terminus of the CS protein in the fusion protein may have thesequence:

(SEQ ID NO: 4) NKNNQGNGQGHNMPNDPNRNVDENANANSAVKNNNNEEPSDKHIKEYLNKIQNSLSTEWSPCSVTCGNGIQVRIKPGSANKPKDELDYANDIEKKICKME KCSSV.

The particle of the invention is sometimes referred to as a virus-likeparticle. It is considered that such particles are more immunogenic thanmonomeric proteins.

In the present invention the particle may comprise no, or substantiallyno, other proteinaceous material.

The particle of the invention may comprise no, or substantially no, freehepatitis B surface antigen protein: that is no, or substantially no,hepatitis B surface antigen protein which is not part of the fusionprotein.

The particle of the invention may comprise no, or substantially no, freeCS protein: that is no, or substantially no, CS protein which is notpart of the fusion protein.

Reference herein to “substantially no” preferably requires the particleto comprise less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or about1% of the particular material referred.

Preferably the particles contain less than 5%, more preferably less than1%, free hepatitis B surface antigen protein.

Preferably, in a particle of the invention at least about 40% or more bymass of the proteinaceous material is derived from Plasmodiumfalciparum.

The ability to have such a high level of Plasmodium falciparum materialin the particles allows a more favourable antibody response with respectto malaria, more specifically a significantly improved antibody responseto Plasmodium falciparum is observed with particles of the invention,and a smaller antibody response is seen to the hepatitis B surfaceantigen.

A reduction in the relative amount of hepatitis B surface antigen in theparticles may also have the advantage that the particles have improvedefficacy in early infancy. If too much hepatitis B surface antigen ispresent there is concern that maternal antibodies present in a younginfant may make the particles less effective as immunogens.

Preferably in a fusion protein of the invention the hepatitis B surfaceantigen is C-terminal to any Plasmodium falciparum material.

The particle may comprise a fusion protein comprising, or consisting of,the sequence of Seq ID No: 1 (R21) or a sequence with at least 80%, 85%,90%, 95%, 98%, 99% or more sequence identity with the sequence of Seq IDNo: 1.

Percentage sequence identity is defined as the percentage of amino acidsin a sequence that are identical with the amino acids in a providedsequence after aligning the sequences and introducing gaps if necessaryto achieve the maximum percent sequence identity. Alignment for thepurpose of determining percent sequence identity can be achieved in manyways that are well known to the man skilled in the art, and include, forexample, using BLAST (National Center for Biotechnology InformationBasic Local Alignment Search Tool).

Variations in percent identity may be due, for example, to amino acidsubstitutions, insertions or deletions. Amino acid substitutions may beconservative in nature, in that the substituted amino acid has similarstructural and/or chemical properties, for example the substitution ofleucine with isoleucine is a conservative substitution.

Preferably a sequence of Seq ID No: 1 includes sequences withconservative substitutions which do not have any significant effect onthe immunogenicity of the resulting fusion protein. Substitutions mayalso be introduced to match better the CS sequence of other strains ofPlasmodium falciparum. The sequence used in the R21 example reportedhere is of the 3D7 strain.

In an alternative embodiment, the particle may comprise, or consist of,a fusion protein having the sequence of Seq ID No: 2 (RTS) or having asequence with at least 80%, 85%, 90%, 95%, 98%, 99% or more sequenceidentity with the sequence of Seq ID No: 2, wherein the particle doesnot comprise any, or substantially any, hepatitis B surface antigenprotein which is not part of the fusion protein.

In another embodiment, the particle may comprise, or consist of, afusion protein having the sequence of Seq ID No: 2 (RTS) or having asequence with at least 80%, 85%, 90%, 95%, 98%, 99% or more sequenceidentity with the sequence of Seq ID No: 2, wherein the particle doesnot comprise any, or substantially any, CS protein which is not part ofthe fusion protein.

Preferably a particle of the invention comprises numerous monomers ofthe fusion protein. The particle may comprise a least 10 fusion proteinmonomers, preferably 20 or more, 30 or more, 40 or more, 50 or more, 60or more, 70 or more, 80 or more, 90 or more, 100 or more fusion proteinmonomers. In one embodiment the particle comprises around 96 fusionprotein monomers.

Preferably a particle of the invention is immunogenic. A particle of theinvention may be capable of eliciting an immune response against themalaria causing parasite Plasmodium falciparum. The immune response maybe therapeutic and/or prophylactic. The immune response may besufficient to reduce or prevent infection or disease cause by Plasmodiumfalciparum. The particle may elicit/produce a protective immune responsewhen administered to a subject, preferably a human subject.

Preferably the immune response elicited by the composition of theinvention affects the ability of Plasmodium falciparum to infect animmunised human. Preferably the ability of Plasmodium falciparum toinfect a human immunised with the composition of the invention isimpeded or prevented. This may be achieved in a number of ways. Theimmune response elicited may recognise and destroy Plasmodiumfalciparum. Alternatively, or additionally, the immune response elicitedmay impede or prevent replication of Plasmodium falciparum.Alternatively, or additionally, the immune response elicited may impedeor prevent Plasmodium falciparum causing disease in the human ornon-human animal. Preferably the immune response elicited is an antibodyresponse.

The particle may be provided in a liquid formulation. Alternatively, theparticle may be provided in a lyophilised form. Alternatively theparticle may be provided in a sugar based formulation dried on membranesas described by Alcock et al. (Sci Transl Med. 2010 Feb. 17;2(19):19ra12).

According to a further aspect the invention provides a method ofproducing particles according to the first aspect of the inventioncomprising expressing the fusion protein in Saccharomyces cerevisiae orPichia pastoris or another methylotrophic yeast such as Hansenulapolymorpha and recovering the fusion protein, preferably in the form ofparticles.

If the fusion protein is expressed in Pichia pastoris, or anothermethylotrophic yeast, expression of the protein may be driven by theAOX1 promoter or by the GAP promoter or by another strong promoter (Vogl& Glieder, New Biotechnology. 2012 Nov. 16. pii: S1871-6784(12)00867-9).

If the fusion protein is expressed in Saccharomyces cerevisiae,expression of the protein may be driven by the TDH3 promoter or byanother strong promoter.

Preferably the fusion protein is expressed at sufficiently high levelsthat upon lysis of the yeast the fusion proteins spontaneouslymultimerise to form particles, sometime referred to as virus-likeparticles.

The DNA encoding the fusion protein may be transiently or constitutivelyexpressed by the yeast. The DNA encoding the fusion protein may beintegrated into the host genome or may be carried on an extracellularcomponent, such as a plasmid. The yeast may contain, 1, 2, 3, 4, 5 ormore copies of the DNA encoding the fusion protein.

The DNA encoding the fusion protein may be codon optimised forexpression in yeast.

A person skilled in the art would be readily able to prepare a suitablehost to express the DNA encoding the fusion protein.

Preferably the Saccharomyces cerevisiae or Pichia pastoris or anothermethylotrophic yeast used in the method of the invention does notexpress any, or any significant, hepatitis B surface antigen proteinwhich is not part of the fusion protein.

Preferably the Saccharomyces cerevisiae or Pichia pastoris or anothermethylotrophic yeast used in the method of the invention does notexpress any, or any significant, CS protein from Plasmodium falciparumwhich is not part of the fusion protein.

The ability to express particles according to the invention in a highyielding yeast strain, such as Pichia pastoris, may simplify and enhancethe biomanufacture of the particles leading to lower cost of goods formanufacture. This saving in cost is particularly important for a malariavaccine which is targeted primarily at populations, especially childrenand infants, in low income countries who require a low cost vaccine.

According to another aspect the invention provides a particle producedby the method of the invention.

According to further aspect the invention provides a host Saccharomycescerevisiae or Pichia pastoris or another methylotrophic yeast cellcomprising a polynucleotide, such as DNA, encoding for the fusionprotein referred to with reference to the particle of the invention.

In one embodiment the invention provides a Saccharomyces cerevisiae orPichia pastoris or another methylotrophic yeast cell comprising DNAencoding a fusion protein comprising, or consisting of, the sequence ofSeq ID No: 1 (R21) or a sequence with at least 80%, 85%, 90%, 95%, 98%,99% or more sequence identity with the sequence of Seq ID No: 1.Preferably expression of the DNA is driven by the AOX 1 promoter.

In another aspect the invention provides a DNA sequence encoding afusion protein referred to with reference to the particle of theinvention. The invention may also provide a vector containing the DNAsequence, wherein the DNA sequence may be operably linked totranscriptional control elements.

According to another aspect the invention provides a compositioncomprising i) a fusion protein comprising at least one NANP repeat (SEQID NO: 7), some or all of the C-terminus of the CS protein fromPlasmodium falciparum and a hepatitis B surface antigen; and ii) arecombinant or non-recombinant viral vector. The viral vector mayexpress an antigen. The viral vector may be a modified vaccinia virusAnkara (MVA). The viral vector may encode TRAP and/or ME-TRAP.Alternatively, or additionally, the antigen expressed by the viralvector may another malaria antigen, such as for example CSP.

The composition may further comprise an adjuvant. The adjuvant maycontain saponin and may be Abisco, matrix M, QS21, AS01 or AS02.

The composition may in part i) comprise the fusion protein as a particleas described herein. Alternatively, or additionally, the composition maycomprise in part i) the fusion protein RTS, and may further comprisefree S antigen from hepatitis B.

According to a still further aspect the invention provides apharmaceutical composition, and in particular a vaccine composition foruse in the prevention of malaria, comprising the particles orcomposition of the invention and a pharmaceutically acceptable carrieror excipient.

Suitable acceptable excipients and carriers will be well known to thoseskilled in the art. These may include solid or liquid carriers. Suitableliquid carriers include water and saline. The proteins of thecomposition may be formulated into an emulsion or they may be formulatedinto biodegradable microspheres or liposomes.

The composition may further comprise an adjuvant. Suitable adjuvantswill be well known to those skilled in the art, and may include Freund'sIncomplete Adjuvant (for use in animals), a saponin derivative, anemulsion such as MF59, and metal salts such as aluminium or calciumsalts, for example alum. The adjuvant may be a squalene-based adjuvant,such as AddaVax (Invivogen) and/or an ISCOM-based adjuvant, such asAbisco/Matrix M (from Isconova), and/or MF59 (Novartis, Siena, Italy).AddaVax™ is based on nano-emulsification of 2 components: Sorbitantrioleate (0.5% w/v) in squalene oil (5% v/v); and Tween 80 (0.5% w/v)in sodium citrate buffer (10 mM, pH 6.5). The nano-emulsion is producedusing a microfluidizer and filtered through a 0.22-μm filter to removelarge droplets and sterilize the final product. The particle size is˜160 nm. MF59 is a ˜160 nm particle emulsion (Novartis, Siena)comprising Squalene: 9.75 mg; Polysorbate 80: 1.175 mg; Sorbitantrioleate: 1.175 mg; Sodium citrate: 0.66 mg; Citric acid: 0.04 mg.Abisco-100 (known as Matrix-M when made to GMP standard) has thefollowing chemical content: purified saponins obtained from a crudeextract of the plant Quillaja saponaria Molina; cholesterol from Lanolinand phosphatidyl choline (phospholipid) from fresh egg yolk; in asuspension of nano-sized (40 nm) cage-like particles consisting of theabove ingredients, in PBS. Matrix M (or Abisco-100) consists of amixture of Matrix A and Matrix C at a ratio of 80:20 to 95:5, preferably85:15. Matrix A leads to T cell induction and has low toxicity, Matrix Cinduces antibodies and has some toxicity. Matrix C contains C fractionof QS separation which corresponds to QS21. Fraction A (in Matrix A)corresponds to QS7.

The composition may also comprise polymers or other agents to controlthe consistency of the composition, and/or to control the release of theantigen/secreted protein from the composition.

The composition may also comprise other agents such as diluents, whichmay include water, saline, glycerol or other suitable alcohols etc;wetting or emulsifying agents; buffering agents; thickening agents forexample cellulose or cellulose derivatives; preservatives; detergents,antimicrobial agents; and the like.

Preferably the active ingredients in the composition are greater than50% pure, usually greater than 80% pure, often greater than 90% pure andmore preferably greater than 95%, 98% or 99% pure. With activeingredients approaching 100% pure, for example about 99.5% pure or about99.9% pure, being used most often.

The composition of the invention may also include in admixture one ormore further antigens. The one or more further antigens may be derivedfrom Plasmodium falciparum or from other species of Plasmodium, such asPlasmodium vivax or Plasmodium malariae.

The pharmaceutical composition or vaccine composition may be provided ina liquid form or in a lyophilised form.

The pharmaceutical composition or vaccine composition may be intendedfor administration in a dose selected to give an appropriate immuneresponse whilst not causing significant side affects. Each dose maycomprise between about 1 and about 1000 μg of fusion protein, forexample between about 1 and about 200 μg of fusion protein, preferablybetween about 1 and about 50 μg of fusion protein. The optimal amount ofparticles/fusion protein to be used can be ascertained by standardstudies well known to a person skilled in the art.

Preferably the pharmaceutical composition or vaccine composition iscapable of producing a protective immune response to Plasmodiumfalciparum.

The phrase “producing a protective immune response” as used herein meansthat the composition is capable of generating a protective response in ahost organism, such as a human or a non-human mammal, to whom it isadministered. Preferably a protective immune response protects againstsubsequent infection or disease caused by Plasmodium falciparum. Theprotective immune response may eliminate or reduce the level ofinfection by reducing replication of Plasmodium falciparum or byaffecting the mode of action of Plasmodium falciparum to reduce disease.

Preferably, if the composition is used as a vaccine, the compositioncomprises an immunologically effective amount of particles according tothe invention. An “immunologically effective amount” of an antigen is anamount that when administered to an individual, either in a single doseor in a series of doses, such as 2 to 4 doses, is effective fortreatment or prevention of infection by Plasmodium falciparum. Thisamount will vary depending upon the health and physical condition of theindividual to be treated and on the antigen. Determination of aneffective amount of an immunogenic or vaccine composition foradministration to an organism is well within the capabilities of thoseskilled in the art.

A composition according to the invention may be for oral, systemic,parenteral, topical, mucosal, intramuscular, intravenous,intraperitoneal, intradermal, subcutaneous, intranasal, intravaginal,intrarectal, transdermal, sublingual, inhalation or aerosoladministration.

The composition may be arranged to be administered as a single dose oras part of a multiple dose schedule. Multiple doses may be administeredas a primary immunisation followed by one or more booster immunisations.Suitable timings between priming and boosting immunisations can beroutinely determined, but will typically be at intervals of 2 weeks to 4months.

Compositions of the invention may be able to induce serum antibodyresponses which mediate the destruction or inactivation of thePlasmodium falciparum after being administered to a subject. Theseresponses are conveniently measured in mice and the results are astandard indicator of vaccine efficacy.

The compositions of the invention may also, or alternatively, be able toelicit an immune response which neutralises Plasmodium falciparum,thereby preventing them from having their normal function and preventingor reducing disease progression without necessarily destroying thePlasmodium falciparum.

A composition according to the invention may be used in isolation, or itmay be combined with one or more other immunogenic or vaccinecompositions, and/or with one or more other therapeutic regimes.

The composition may be intended for administration with a viral vector.The viral vector may encode one or more pre-erythrocytic malariaantigens or antigens from other stages of the malaria parasite'slife-cycle. The antigen encoded may be the malarial antigen TRAP(thrombospondin related adhesion protein) or ME-TRAP. The strong T cellimmunogenicity of such vectored vaccines that are known to have somepartial efficacy against the liver-stage of malaria infection, is eithermaintained or even, surprisingly, enhanced when combined with acomposition of the invention. The invention may provide a combinationmalaria vaccine, comprising particles according to the invention and aviral vector. The combination would have potent anti-sporozoite activityprimarily induced by the particles and anti-liver-stage activityprimarily induced by the viral vectors.

The composition may be intended to be administered with one or more ofan adenovirus vector and an MVA vector. The composition may beadministered in a prime boost regimen, wherein the prime comprises theparticles of the invention and an adenovirus vector or an MVA vector,and the boost comprises the particles of the invention and adenovirus oran MVA vector. Preferably the prime comprises an adenovirus and boostcomprises an MVA vector. Preferably the prime and the boost include anadjuvant with the particles. The adenoviral vector or the MVA viralvectors and the particles of the invention may be administered as amixture, or alternatively they may be administered separately, meaningat separate immunisation sites or at separate time points. Ifadministered separately the vector and particles may be administered atthe same or different sites. The administration of vector and particlesmay be simultaneous, or substantially simultaneous, for example, within10 minutes of each other, or the administration may be sequential.Preferably if the vector and particles are administered sequentially,they are administered within at most about 30 days, 7 days, 6 days, 4days, 2 days, 24 hours or less, of each other.

The composition of the invention may further comprise one or more viralvectors.

According to yet another aspect the invention provides a fusion proteincomprising the sequence of Seq ID No: 1 (R21) or comprising, orconsisting of, a sequence with at least 80%, 85%, 90%, 95%, 98%, 99% ormore sequence identity with the sequence of Seq ID No: 1. Preferably thefusion protein consists of the sequence of Seq ID No: 1 (R21).

According to a further aspect the invention provides a method oftreating a subject susceptible to Plasmodium falciparum infectioncomprising administering to the subject an effective amount of apharmaceutical composition or vaccine composition according to theinvention.

According to a further aspect the invention provides a method ofimmunising a subject against malaria/Plasmodium falciparum infectioncomprising administering to the subject an effective amount of apharmaceutical composition or vaccine composition according to theinvention.

According to another aspect the invention provides a use of apharmaceutical composition or vaccine composition according to theinvention in the preparation of a medicament for thetreatment/immunisation of a subject susceptible to Plasmodium falciparuminfection.

According to another aspect the invention provides a pharmaceuticalcomposition or vaccine composition according to the invention for use inthe treatment/immunisation of a subject susceptible to Plasmodiumfalciparum infection.

According to further aspect the invention provides a kit for use ininducing an immune response to Plasmodium falciparum comprising aparticle or composition according to the invention and instructionsrelating to administration. The kit may further comprise one or more ofan adjuvant and a viral vector. If an adjuvant is present preferably itis in the same composition as the particle. The viral vector may be in asame of different composition to the particle.

The skilled man will appreciate that any of the preferable featuresdiscussed above can be applied to any of the aspects of the invention.

Preferred embodiments of the present invention will now be described,merely by way of example, with reference to the following figures andexamples.

FIG. 1—provides the sequence of R21 (Seq ID No: 1) and RTS (Seq ID No:2);

FIG. 2A and FIG. 2B—shows the results of the analysis of purified R21particles by transmission electron microscopy—FIG. 2A. In FIG. 2Bparticles are shown that lack the 105 amino acid of the CS protein Cterminus. The particles are negatively stained with 2% uranyl acetate;

FIG. 3—shows the results of the analysis of R21 purified particles byreducing gel electrophoresis. FIG. 3A shows silver stained R21 purifiedparticles. FIG. 3B shows the results of a western blot using monoclonalanti-NANP antibody (“NANP” disclosed as SEQ ID NO: 7; from MR4, named2A10).

FIG. 4—shows the results of immunisation studies. BALB/c mice wereimmunised intramuscularly with 0.5 μg R21 with either Alhydrogel (85 ug)or Abisco-100 (12 ug) or AddaVax. Three shots were given 3 weeks apartand NANP-specific antibody (“NANP” disclosed as SEQ ID NO: 7) responseswere assayed by ELISA 3 weeks after each immunisation. Median responsesare shown. “NANP₆C” disclosed as SEQ ID NO: 12.

FIG. 5—demonstrates that R21 in Abisco adjuvant induced high titreantibodies to the NANP repeat (SEQ ID NO: 7) of the CS protein butmodest antibody titres to HBsAg. BALB/c mice were immunisedintramuscularly with 5 μg R21 with Abisco-100. Three shots were given 3weeks apart and HBsAg-specific antibody responses were assayed by ELISA3 weeks after the 3rd immunisation. Median responses are shown.

FIG. 6—details the results of IFN-γ Spleen ELISpot analysis for BALB/cmice immunised intramuscularly with 0.5 μg R21 with either Alhydrogel(85 ug) or Abisco-100 (12 ug) or AddaVax. Three shots were given 3 weeksapart and NANP-specific antibody (“NANP” disclosed as SEQ ID NO: 7)responses were assayed by ELISA 3 weeks after each immunisation. Medianresponses are shown.

FIG. 7—demonstrates that R21 in adjuvant can be combined with viralvectors without impairing humoral immunogenicity. BALB/c mice wereimmunised with either 2 shots of R21+Abisco-100 or the ChAd63 (Ad) METRAP—MVA ME TRAP 8 week prime-boost regime alone or combined together,as detailed in Table 1. Humoral responses were assayed in the NANP (SEQID NO: 7) ELISA carried out 3 weeks and 8 weeks after the firstimmunisation, and 3 weeks after the final immunisation. Median responsesare shown. No impairment of the humoral immunogenicity of R21 isobserved. “NANP₆C” disclosed as SEQ ID NO: 12.

FIG. 8—demonstrates that R21 in adjuvant can be combined with viralvectors without impairing T cell immunogenicity. BALB/c mice wereimmunised with either 2 shots of R21+Abisco-100 or the ChAd63 (Ad) METRAP—MVA ME TRAP 8 week prime-boost regime alone or combined together.Cellular responses were assayed in an IFN-γ spleen ELISpot to a pool ofCS peptides (A) or the Pb9 peptide (B) 3 weeks after the finalimmunisation. Median responses are shown. No impairment of the T cellimmunogenicity of R21 or the viral vectors is observed.

FIG. 9—demonstrates that mixing R21 plus adjuvants with vectors canenhance antibody responses to the vector encoded antigen. BALB/c micewere immunised intramuscularly with the ChAd63 (Ad) ME TRAP—MVA ME TRAP8 week prime-boost regime alone or combined with either Abisco-100 orAddaVax. TRAP-specific antibody responses were assayed by ELISA 3 weeksand 8 weeks after the first immunisation and 3 weeks after the secondMedian responses are shown.

FIG. 10—demonstrates that the R21 in a ISCOM adjuvant, such as Abisco orMatrix M (available from Isconova, Uppsala, Sweden) provides high levelprotective efficacy against malaria sporozoite infection in mice. Themice were infected at two immunisations with the R21 particle inadjuvant by a transgenic Plasmodium berghei parasite, transgenic for theP. falciparum CS protein gene. Such parasites are described in theresearch literature (Tewari R et al. J Biol Chem. 2002 Dec. 6;277(49):47613-8; Kaba S et al. PLoS One. 2012; 7(10):e48304). Theresults demonstrate that the R21 particle not only shows excellentimmunogenicity against P. falciparum CS but potent efficacy in a veryrelevant malaria infection model. IM: intramuscular.

FIG. 11—this table illustrates the study design used to consider theimmunogenicity of R21 without an adjuvant.

FIG. 12—demonstrates CS-specific IgG responses to R21 with differentadjuvants. BALB/c mice were immunised intramuscularly with 0.5 ug R21alone or formulated with Alhydrogel or Abisco-100. Three immunisationswere given three weeks apart and the NANP-specific antibody (“NANP”disclosed as SEQ ID NO: 7) titres were measured by ELISA 3 weeks aftereach immunisation. Median responses are shown.

FIG. 13—demonstrates IFN-γ Spleen ELISpot responses to R21 with MatrixM. BALB/c mice were immunised intramuscularly with 0.5 μg R21 withMatrix M (12 ug). Three shots were given 3 weeks apart and CS-specific Tcell responses were measured in the spleen by IFN-γ ELISpot 3 weeksafter the final immunisation. Median responses are shown.

FIG. 14—illustrates the study design for a vaccine immunisation intervalstudy. The illustrated vaccine regimens were used to assess theimmunogenicity of R21+Matrix M in BALB/c mice

FIG. 15—demonstrates CS-Specific IgG responses to R21 and Matrix M.BALB/c mice were immunised intramuscularly with 0.5 μg R21 with Matrix M(12 ug). NANP-specific antibody (“NANP” disclosed as SEQ ID NO: 7)titres were assayed by ELISA 3 weeks after each immunisation. Medianresponses are shown.

FIG. 16—demonstrates IFN-γ Spleen ELISpot responses to R21 and Matrix M.BALB/c mice were immunised intramuscularly with 0.5 μg R21 with Matrix M(12 ug). Three shots were given 3 weeks apart and CS-specific T cellresponses were measured in the spleen by IFNg ELISpot 3 weeks after thefinal immunisation. Median responses are shown.

FIG. 17—shows the study design used to compare R21 and the CSPrecombinant protein

FIG. 18—demonstrates CS-specific IgG responses to R21 or CSP with eitherMatrix M, MF59 or L-AS01. BALB/c mice were immunised intramuscularlywith 0.5 μg R21 or CSP with either Matrix M, MF59 or L-AS01.NANP-specific antibody (“NANP” disclosed as SEQ ID NO: 7) titres wereassayed by ELISA 3 weeks after each immunisation. Mean responses with SDare shown (Groups compared by One-way ANOVA with Bonferroni's post-test.ns=non-significant, *p<0.05, **p<0.01, ***p<0.001).

FIG. 19—demonstrates CS-specific T cells to R21 or CSP with eitherMatrix M, MF59 or L-AS01. CS-specific T cells. BALB/c mice wereimmunised intramuscularly with 0.5 μg R21 or CSP with either Matrix M,MF59 or L-AS01. CS specific T cell responses were measured in the bloodby ICS and flow cytometry, 2 weeks after the boost vaccination. IsolatedPBMC's were re-stimulated with a pool of CS peptides and frequencies ofcytokine secreting CD4+ T cells were measured (IFNg, TNF and IL2). Meanresponses shown.

FIG. 20—illustrates an R21 vs CSP challenge experiment. BALB/c mice wereimmunised intramuscularly with two shots of 0.5 μg R21 or CSP withMatrix M, 8 weeks apart. A) Mice were challenged 3 weeks after boostwith 1000 sporozoites injected i.v. and time to 1% parasitemia wasdetermined by thin film blood smear from day 5 post challenge. Mice weresterilely protected if they had no blood stage parasites by day 14.Survival curves compared by Log-rank (Mantel-Cox) Test. B) NANP-specificantibody (“NANP” disclosed as SEQ ID NO: 7) titres were assayed by ELISA3 weeks after each immunisation. Mean responses with SD are shown(Groups compared by One-way ANOVA with Bonferroni's post-test. **p<0.01,***p<0.001). C) T cell responses were measured in the blood by ICS andflow cytometry, 2 weeks after the boost vaccination. Isolated PBMC'swere re-stimulated with a pool of CS peptides and frequencies ofcytokine secreting CD4+ T cells were measured secreting (IFNg, TNF andIL2). Mean responses with SEM are shown.

FIG. 21—demonstrates the enhanced efficacy of combined immunisation withR21 and viral vectors. BALB/c mice were immunised intramuscularly withR21+MF59 or the ChAd63 PbTRAP-MVA PbTRAP regimen alone (A or B) orcombined together (C and D). Mice were challenged 3 weeks after boostwith 1000 sporozoites injected i.v. and time to 1% parasitemia wasdetermined by thin film blood smear from day 5 post challenge. Mice weresterilely protected if they had no blood stage parasites by day 14.Survival curves were compared by Log-rank (Mantel-Cox) Test.

FIG. 22—demonstrates the efficacy of IgG passive transfer. BALB/c micewere immunised intramuscularly with R21+Matrix M twice, 8 weeks apart,mice were bled and total IgG was purified. Naïve mice were immunisedwith either B) 150 ug C) 750 ug or D) 1.5 mg of total IgG fromvaccinated mice i.v. A control group also received IgG from unvaccinatedmice A). All mice were challenged 5 hours after IgG transfer with 1000sporozoites injected i.v. and time to 1% parasitemia was determined bythin film blood smear from day 5 post challenge. Mice were sterilelyprotected if they had no blood stage parasites by day 14. Survivalcurves were compared by Log-rank (Mantel-Cox) Test.

FIG. 23—demonstrates that NANP-specific IgG (“NANP” disclosed as SEQ IDNO: 7) correlates with efficacy. Groups of BALB/c mice receivedincreasing doses of total IgG from vaccinated mice (R21+Matrix M) andhad moderate—good levels of NANP-specific IgG (“NANP” disclosed as SEQID NO: 7) titres at the time of challenge (A). The level ofNANP-specific IgG (“NANP” disclosed as SEQ ID NO: 7) correlated with thetime taken for mice to develop 1% parasitemia (B) (correlation testedusing Pearsons correlation, p=0.0008, 1=0.659).

In order to exemplify the invention described herein particlescomprising the fusion protein R21 (Seq ID no: 1, FIG. 1) were producedand their immunogenic properties were considered.

Development of R21 Expressing Yeast Materials and Methods R21 ExpressionPlasmid

A sequence which encodes for the fusion protein R21 (Seq ID No: 1 andFIG. 1) of was cloned into the pPink-HC expression plasmid from thePichiaPink™ Expression System (Invitrogen, Cat. no. A11150).

The R21 protein comprises the Hepatitis B surface antigen (HBsAg) adwserotype and a C-terminal portion of the circumsporozoite (CS) proteinof the Plasmodium falciparum strain NF54. The sequence comprises 410amino acids from N to C terminus:

75 amino acids of the NANP repeat (“NANP” disclosed as SEQ ID NO: 7)from the CS protein comprising MDP followed by 18 NANP repeats (“18 NANPrepeats” disclosed as SEQ ID NO: 9):MDPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANP (SEQ ID NO: 3)105 amino acid of the CS protein C terminus:

(SEQ ID NO: 4) NKNNQGNGQGHNMPNDPNRNVDENANANSAVKNNNNEEPSDKHIKEYLNKIQNSLSTEWSPCSVTCGNGIQVRIKPGSANKPKDELDYANDIEKKICKME KCSSV(The 10 C terminal amino acids (FNVVNSSIGL (SEQ ID NO: 10)) of the CSprotein have been removed.)4 amino acids from the pre-S2 region of the hepatitis B virus:

(SEQ ID NO: 11) PVTN followed by 226 amino acids of the HBsAg adw serotype:

(SEQ ID NO: 5) MENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGSPVCLGQNSQSPTSNHSPTSCPPICPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLIPGSTTTNTGPCKTCTTPAQGNSMFPSCCCTKPTDGNCTCIPIPSSWAFAKYLWEWASVRFSWLSLLVPFVQWFVGLSPTVWLSAIWMMWYWGPSLYSIVSPFIPLLPIFFCLWVYI

R21 Expressing Yeast

PichiaPink™ Strain 4 (Invitrogen, Cat. no. A11150), which is a doubleknock-out for proteinases A and B (i.e. pep4 and prb1), was transformedby electroporation with the pPink-HC expression plasmid encoding R21.Positively transformed colonies were selected by growth on adeninedeficient agar plates.

Initial Characterisation of Transformed Yeast

Positive colonies were selected and grown in small 1 mL pilot expressioncultures and protein expression induced by the addition of methanol.Levels of protein expression in each clone were then analysed by westernblot. Induced R21 expressing yeast samples were disrupted in laemmlilysis buffer and western blot analysis was performed using an antibodyto the NANP repeat (SEQ ID NO: 7) in the CSP portion of the fusionprotein, (monoclonal anti-NANP antibody (“NANP” disclosed as SEQ ID NO:7) (MR4, 2A10)) and a monoclonal anti-HBsAg antibody (Serotec, MCA4658).Expression of the fusion protein that was recognised by both antibodiesat the correct size was confirmed.

Growth of Pichia Pastoris and Induction of R21 Expression

One R21 expressing clone was selected and grown in a 1 L batch culturewith BMGY (Buffered complex medium containing glycerol). R21 expressionwas induced by the addition of methanol by changing the media to BMMY(Buffered complex medium containing methanol). Expression was inducedwith 0.5% methanol for 3 days at which point the yeast was pelleted bycentrifugation at 1,500×g for 5 minutes, the supernatant removed and theyeast frozen at −80° C.

Extraction and Purification of R21 Cell Disruption

Yeast pellets were thawed on ice and resuspended in a lysis buffercontaining 10 mM Tris (pH 7.8), 0.1% Triton X-100, 1 mM EDTA. Acidwashed glass beads (0.425-600 um) were added and the sample wasdisrupted by 10 cycles of vortexing for one minute then placing on icefor one minute.

Purification Clarification

Before purification the yeast debris was removed by centrifugation for 5minutes at 1,500×g and this lysate was then clarified byultracentrifugation at 13,000×g for 20 minutes.

CsCl Discontinuous Gradient Centrifugation

The clarified lysate was then layered onto a discontinuous CsCl gradientcontaining layers of 1.3 g/ml CsCl and 1.1 g/ml CsCl. Afterultracentrifugation for 2 hours at 41,000 rpm in a SW41 Ti rotor,(Beckman Coulter Optima L-100 XP) the particle containing fraction wascollected.

Gel Filtration

The particle containing fraction was then applied to a PD10 columncontaining Sephedex G100. The sample was eluted in 10 mM Tris (pH7.8)and particle containing fractions were collected and pooled.

CsCl Isopycnic Gradient Centrifugation

The pooled sample was then added to an isopycnic CsCl gradientcontaining 1.2 g/ml CsCl. After ultracentrifugation for 20 hours at41,000 rpm in a SW41 Ti rotor, (Beckman Coulter Optima L-100 XP) theparticle containing fraction was collected.

Size Exclusion Chromatography on Sephacryl 500

The sample was then applied to a Hiprep 16/60 Sephacryl S-500 HR gelfiltration column (GE Healthcare) to exchange the buffer and remove anyremaining lower molecular weight contaminants. The sample was eluted in10 mM Tris buffer (pH7.8) and pure particle containing fractions werepooled.

R21 Characterisation Electron Microscopy

To confirm the presence and size of particles, the purified R21 particlepreparation was analysed by negative staining with 2% uranyl acetate ona transmission electron microscope (FIG. 2A). Particles appear to beapproximately 22 nm in size.

Particles were also made that lacked the 105 amino acid of the CSprotein C terminus, these are depicted in FIG. 2B.

Gel Electrophoresis; Silver Stain and Western Blot Analysis

The purified R21 particle preparation was analysed by reducing gelelectrophoresis. The sample was silver stained to assess the purity andanalysed by western blotting with a monoclonal anti-NANP antibody(“NANP” disclosed as SEQ ID NO: 7; MR4, 2A10) and an anti-HBsAgmonoclonal antibody to assess the immunoreactivity of the product. Thesame band was recognised by both antibodies and the purified product wasgreater than 90% pure based on the silver stained gel (FIG. 3).

ELISA to CS Repeat Region

The presence and accessibility of the NANP repeat (SEQ ID NO: 7) regionon the surface of the particle was assessed by sandwich ELISA using twoantibodies to the NANP repeat (“NANP” disclosed as SEQ ID NO: 7) region.Maxisorb 96 well plates (Nunc) were coated with mouse monoclonalanti-NANP antibody (“NANP” disclosed as SEQ ID NO: 7; MR4, 2A10),incubated with purified R21 particle and a rabbit polyclonal serum (MR4,MRA-24) was used for detection. The purified R21 particle prep gave astrong positive signal.

ELISA to CS Repeat Region and HBsAg

The presence and accessibility of the HBsAg portion of fusion protein inthe particle was assessed by sandwich ELISA using a monoclonal antibodyto the NANP repeat (SEQ ID NO: 7) region (MR4, 2A10) and a cocktail ofantibodies to the HBsAg from Monolisa ULTRA HBsAg ELISA kit (Biorad).The purified R21 particle prep gave a weak positive signal thusconfirming the presence and accessibility of the HBsAg epitopes on thesame NANP (SEQ ID NO: 7) containing R21 particles.

Quantification by Absorbance at 280 nm

The purified particle prep is quantified by measuring absorbance at 280nm.

Larger Scale Manufacturing of R21

To assess the feasibility of larger scale manufacturing a new processwas developed in a GMP facility approved by the UK Medicines andHealthcare products Regulatory Agency (MHRA), the ClinicalBiomanufacturing Facility at the University of Oxford. This large scaleprocess provided satisfactory yields of R21 particles at a larger scalethat were immunogenic in small animals, indicating that a GMP compatibleprocess is achievable for clinical grade manufacturing of the R21particle immunogen.

Immunogenicity of R21 Particles in Mice

Immunogenicity was assessed in mice by measuring the antibodiesgenerated to the NANP repeat (SEQ ID NO: 7) in an ELISA and by measuringT cells specific for a pool of CS peptides contained within the R21vaccine by IFN-γ spleen ELISpot. The antibody response to the HBsAg wasalso assessed in an ELISA.

Anti-NANP Antibody ELISA (“NANP” Disclosed as SEQ ID NO: 7)

Anti-CSP antibodies induced by R21 immunisation were assessed in anELISA using NANP₆C peptide (SEQ ID NO: 12). The NANP₆C peptide (SEQ IDNO: 12) consists of 6 copies of the NANP repeat followed by a C.Maxisorb (SEQ ID NO: 12). 96 well plates (Nunc) were coated with thisantigen and incubated with serum samples using a 3 fold serial dilutionstarting at a dilution of 1:1000. Mouse antibodies were detected withalkaline phosphatase conjugated anti-mouse IgG and pNPP (p-NitrophenylPhosphate, Disodium Salt) substrate and absorbance read at 405 nm. Theresults are expressed as endpoint titre which is defined as the dilutionat which the OD of the sample is equal to background.

The R21 particle vaccine was found to be highly immunogenic in mice andthere is a notable boost effect after each vaccination (FIG. 4.).Furthermore, if R21 is administered with the adjuvant Abisco or AddaVaxslightly higher antibody titres were induced as compared to R21administered with Alhydrogel.

IFN-γ Spleen ELISpot

T cells induced by immunisation with R21 were measured using IFN-γELISpot assay. Fresh splenocytes were isolated and incubated induplicate wells for 20 hours. Cells were restimulated with a pool of CSpeptides spanning the entire CS protein at a final concentration of 2ug/ml. Plates were coated and INF-γ was detected using antibodies fromMabtech. Spots were developed using an alkaline phosphatase substratekit from Biorad and counted using an ELISPOT counter (AID). The resultsare presented in FIG. 6 and are expressed as number of spot formingcells (SFC) per million splenocytes.

T cell responses were only measured after the third vaccination. R21administered with Abisco is more effective at inducing T cells to the CSpeptides in mice than R21 administered with Ahydrogel.

Anti-HBsAg Antibody ELISA

Anti-HBsAg antibodies induced by R21 immunisation with Abisco adjuvantwere assessed in an ELISA using HBsAg particle. The HBsAg particle wascoated onto maxisorb 96 well plates (Nunc) and the plates were incubatedwith serum samples using a 3 fold serial dilution starting at a dilutionof 1:100. Mouse IgG antibodies were detected and the results expressedas for the anti-NANP antibody ELISA (“NANP” disclosed as SEQ ID NO: 7)above.

HBsAg antibodies were assessed after three immunisations. Low antibodytitres to the HBsAg were detected in all of the immunised mice incontrast to high titre antibodies against NANP (SEQ ID NO: 7; FIG. 5).This result shows that the R21 particle induces preferentiallyantibodies to the malaria rather than the hepatitis B component of R21.

Further experimentation supported the finding discussed above and inparticular demonstrated that R21, when administered alone, is able toinduce moderate CS-specific antibodies and T cells after 3 low doseimmunisation. More specifically, groups of BALB/c mice were immunisedintramuscularly with R21 alone or formulated with adjuvant as detailedin FIG. 11. Three immunisations were given three weeks apart and theimmunogenicity was assessed by measuring serum antibody titres 3 weeksafter each immunisation and antigen-specific T cell responses in thespleen 3 weeks after the final immunisation. After the thirdimmunisation the CS-specific IgG titres (FIG. 12) and CS-specific Tcells (FIG. 13) were not different between the groups receiving R21alone or R21+Alhydrogel. These results demonstrate that R21 isimmunogenic alone, and in the absence of an adjuvant can stimulatecellular immunity, and can still induce low levels of CS-specific IFN-γproducing T cell on its own.

Immunogenicity of R21 Particles with Viral Vector Vaccines in Mice

The immunogenicity of mixtures of R21 plus adjuvant with adenoviral andMVA viral vectors expressing the ME TRAP antigen (O'Hara et al J InfectDis 2012) were assessed in BALB/c mice as detailed in Table 1. Noimpairment of humoral (FIG. 7) or T cell immunogenicity (FIG. 8) wasobserved with the mixtures. This is an important result as it indicatesthat despite the potential negative impact of an adjuvant on theimmunogenicity of viral vectored vaccines, no negative impact wasobserved. This is surprising as many other adjuvants other than thesaponin and emulsion types adjuvants used in this work have been foundto negatively impact on the immunogenicity of viral vectors. Thisobservation has potential utility as it could allow these two types ofmalaria vaccine, R21 which targets sporozoites, and viral vectorsencoding the TRAP antigen, which target the liver-stage of malaria, tobe combined successfully.

In particular a combination of R21, or a similar RTS or RTS,S likeparticle, plus a saponin containing adjuvant such as Abisco, matrix M,QS21, AS01 or AS02, plus MVA encoding TRAP or ME TRAP were identified asa novel and particularly preferred vaccine combination for malaria.FIGS. 7 and 8 show that this combination allows both exceptionally highantibody titres (to CSP) and T cell responses to TRAP to be attained. Asthese are arguably the best characterised correlates of protectiveimmunity to pre-erythrocytic malaria in humans the use of thiscombination vaccine for boosting malaria immunity should be particularlyeffective. It is emphasized that MVA is a vector that allows multipleantigens to be expressed so the MVA expressing TRAP in this mixture mayalso express another malaria antigens, such as for example CSP, from thesame or another MVA locus, or may even express an antigen from anotherpathogen, such as antigen 85A from Mycobacterium tuberculosis. Thiscombination vaccine with an MVA expressing antigens from Mycobacteriumtuberculosis as well as Plasmodium falciparum, along with R21 (or asimilar particle such as RTS or RTS,S) plus a saponin adjuvant (such asmatrix M or Iscomatrix or AS01 or AS02) or an emulsion adjuvant, such asAddavax or MF59, could therefore boost immunity to both tuberculosis andmalaria.

TABLE 1 Vaccine co-administration study Vaccine regimens used to assessthe immunogenicity of R21 + adjuvant or ChAd63 ME TRAP- MVA ME TRAP inBALB/c mice administered alone or in combination. Combination vaccinesformulated together and administered in the same syringe. No. Gp miceParticle + Adjuvant No. shots Viral vector Interval 1 6 0.5 μg R21 +Abisco IM 2 — 8 weeks 2 6 — — 1 × 10{circumflex over ( )}8 ChAd63 METRAP IM 8 weeks 1 × 10{circumflex over ( )}6 MVA ME TRAP IM 3 6 0.5 μgR21 + Abisco IM 2 1 × 10{circumflex over ( )}8 ChAd63 ME TRAP IM 8 weeks1 × 10{circumflex over ( )}6 MVA ME TRAP IMImmunogenicity of Viral Vector Insert with Adjuvants

On mixing R21 plus two adjuvants with the same adenoviral and MVA viralvectors an increased antibody response to the encoded antigen, TRAP, wassurprisingly observed (FIG. 9). This was found whether or not the R21particle was included in the mixture, indicating that both the saponinAbisco and the squalene-based emulsion Addavax can enhance antibodyresponses to a viral vector encoded insert. A variety of similaradjuvants such as QS21 formulations (like Abisco) and MF59 (likeAddavax) are available and so may be used to enhance antibody responsesto a viral vector encoded antigen.

Anti-TRAP Antibody ELISA

Anti-TRAP antibodies induced by viral vector immunisation were assessedin an ELISA using TRAP recombinant protein. The TRAP protein was coatedonto maxisorb 96 well plates (Nunc) and the plates were incubated withserum samples using a 3 fold serial dilution starting at a dilution of1:100. Mouse IgG antibodies were detected and the results expressed asfor the anti-NANP antibody ELISA (“NANP” disclosed as SEQ ID NO: 7)above.

R21 can be Administered in a Number of Regimens for OptimalImmunogenicity

Groups of BALB/c mice were immunised with 4 different regimens asdescribed in FIG. 14 with R21+Abisco-100, CS-specific antibodies weremeasured 3 weeks after each immunisation and CS-specific T cells weremeasured 3 weeks after the final immunisation FIGS. 15 & 16respectively. After the final immunisations there was no differencebetween any groups in the level of CS-specific antibodies or T cells.This indicates that high titres antibodies and moderate levels of Tcells can be generated with multiple regimens so interval will notaffect the level of antibodies induced when R21 is combined with viralvectors.

R21 is More Immunogenic than CSP Recombinant Protein

Immunisation with R21 in a range of adjuvants induces higher antibodytitres and CS-specific T cells than CSP recombinant protein. A studyrecently compared four CSP protein vaccines head to head to determinewhich elicited the highest immune responses and superior efficacy. Themost immunogenic recombinant protein evaluated was produced in E. coliby Gennova (Pune, India) and this was obtained from PATH Malaria VaccineInitiative (MVI, Washington D.C.) for assessment against R21. Thisassessment was performed with 3 different adjuvants Matrix M (similar toAbisco-100), MF59 (similar to AddaVax) and a biosimilar of theproprietary GSK adjuvant AS01, obtained from Lausanne here calledL-AS01.

Groups of BALB/c mice were immunised twice, 8 weeks apart with 0.5 ug oreither R21 or CSP formulated with 3 different adjuvants as detailed inFIG. 17. When comparing the two groups that received the same adjuvant,after prime the anti-NANP IgG (“NANP” disclosed as SEQ ID NO: 7)responses were higher in all groups receiving R21 compared to the groupsreceiving CSP, between, 2.6-4.7 fold higher (FIG. 18-A). The same trendwas seen after boost at week 11, with the responses being between 4.5-22fold greater in the R21 groups (FIG. 18-B). There was also nosignificant difference between the responses induced by the 3 adjuvantsfor either R21 or CSP, at any time point. Therefore if the data arepooled together for the R21 groups or the CSP groups at each time pointthe responses to the particle vaccine, R21 are significantly higher atall time-points than the recombinant protein in the 8 week prime boostregimen (FIG. 18-C). CS-specific T cell responses were also measured andmice immunised with the adjuvants Matrix M or L-AS01 adjuvants developedhigher frequencies of T cells than those immunised with MF59 and thoseimmunised with R21 induced higher frequencies of T cells than CSP (FIG.19).

R21 is More Efficacious than CSP Recombinant Protein

R21 is not only more immunogenic but also more efficacious than CSPrecombinant protein. Mice were immunised with R21 or CSP formulated withMatrix M and challenged with transgenic parasites (P. berghei transgenicfor P. falciparum CSP) 3 weeks after boost. R21+Matrix M protected 87.5%of the vaccinated mice and CSP+Matrix M protected only 42.5% (FIG.20-A). The levels of both CS-specific antibodies and T cells were alsohigher in the R21+Matrix M groups (FIG. 20-B+C).

Protective Efficacy of R21 Particles Against Malaria Sporozite Infectionin a Mouse Model

34 Balb/c mice were divided into four groups. Group 1 was immunised with0.5 micrograms of R21 in Abisco adjuvant intramuscularly, twice with aneight-week interval. Group 2 received the same dose of R21 with the verysimilar matrix M adjuvant (also supplied by Isconova, Uppsala, Sweden)with the same interval; the Matrix M is suitable for clinical use. Group3 received a single dose of 1×108 ifu of a chimpanzee adenovirus(ChAd63) expressing the CS gene followed 8 weeks later by a single doseof 1×106 pfu of an MVA vector expressing the CS gene, both by theintramuscular route. Group 4 were immunised with adjuvant only twicewith an 8 week interval, 5 mice receiving Abisco and 5 receiving MatrixM. Three weeks after the day of the booster immunisation all mice wereinfected by a 1000 sporozoites intravenously of a P. berghei parasitetransgenic for the circumsporozoite gene of P. falciparum. Mice werescored for time to 1% parasitaemia by microscopy. 10/10 control adjuvantonly mice were infected; 6/8 from Group 3 were infected showing modestefficacy with the vectored vaccines. In contrast only 1/16 mice inGroups 1 and 2 were infected showing 94% sterile efficacy with R21 withthe ISCOM adjuvant formulations.

Vaccine Efficacy is Enhanced by Combining R21 and Viral Vectors

Immunisation with either R21+MF59 or the ChAd63 PbTRAP−MVA PbTRAPregimen was protective against sporozoite challenge in BALB/c miceeliciting a significant delay in the time taken to develop 1%parasitemia in the blood (FIGS. 21-A and B). Combining these two vaccineregimens together resulted in an increase in the efficacy of bothvaccine regimens (FIGS. 21-C and D).

Vaccine Efficacy is Enhanced by Combining R21 and Viral Vectors

Groups of mice receiving a passive transfer of increasing doses of totalIgG from mice immunised with R21+Matrix M were protected againstsporozoite challenge in a dose dependant manner (FIG. 22). NANP-specificIgG (“NANP” disclosed as SEQ ID NO: 7) titres were measured 1 day afterchallenge (FIG. 23-A) and mice had good to moderate titres thatcorrelated with the efficacy measured as delay to 1% parasitemia (FIG.23-B).

1. A method of immunizing a subject against malaria/Plasmodiumfalciparum infection comprising administering to the subject aneffective amount of a pharmaceutical composition or vaccine composition,the composition comprising (i) a fusion protein comprising at least oneNANP repeat (SEQ ID NO: 7), some or all of the C-terminus of the CSprotein from Plasmodium falciparum and a hepatitis B surface antigen;and optionally (ii) a recombinant or non-recombinant modified vacciniavirus Ankara (MVA); and (iii) a pharmaceutically acceptable carrier orexcipient.
 2. The method of claim 1, wherein the fusion protein and theMVA are administered separately, simultaneously, or sequentially.
 3. Acomposition comprising (i) a fusion protein comprising at least one NANPrepeat (SEQ ID NO: 7), the C-terminus of the CS protein from Plasmodiumfalciparum comprising the sequence:NKNNQGNGQGHNMPNDPNRNVDENANANSAVKNNNNEEPSDKIEKEYLNKIQNSLSTEWSPCSVTCGNGIQVRIKPGSANKPKDELDYANDIEKKICKMEKCSSVFN VVNSSIGI (SEQ ID NO:6); or the sequence NKNNQGNGQGHNMPNDPNRNVDENANANSAVKNNNNEEPSDKHIKEYLNKIQNSLSTEWSPCSVTCGNGIQVRIKPGSANKPKDELDYANDIEKKICKMEKCSSV (SEQ ID NO: 4), ora sequence with 90% or more sequence identity therewith, and a hepatitisB surface antigen (HBsAg); and optionally (ii) a recombinant ornon-recombinant modified vaccinia virus Ankara (MVA).
 4. The compositionof claim 3, wherein the fusion protein is provided as a virus-likeparticle, the virus-like particle comprising no free HBsAg.
 5. Thecomposition of claim 3, wherein the fusion protein is RTS.
 6. Thecomposition of claim 5, further comprising free HBsAg.
 7. Thecomposition of claim 3, wherein the composition is provided as apharmaceutical composition, or a vaccine composition, for use in theprevention of malaria, the composition further comprising apharmaceutically acceptable carrier or excipient.
 8. The composition ofclaim 7, further comprising an adjuvant.
 9. The composition of claim 7,further comprising one or more further antigens.
 10. The composition ofclaim 7, wherein the composition is capable of producing a protectiveimmune response to Plasmodium falciparum.
 11. The composition of claim7, further comprising one or more viral vectors.
 12. The method of claim3, wherein the sequence with 90% or more sequence identity therewithcomprises only conservative substitutions or substitutions introduced tomatch the sequence of a strain of Plasmodium falciparum other thanstrain 3D7.